1. TECHNICAL FIELD
The present inventions relates to methods of transplanting cells. More specifically, the present invention relates to methods of transplanting cells to create a localized immunosuppressive effect in the tissue receiving the transplanted cells.
2. BACKGROUND ART
The central nervous system (CNS) has poor regenerative capacity which is exemplified in a number of neurodegenerative disorders. An example of such a disorder is Parkinson""s disease. The prefened pharmacotherapy for Parkinson""s disease is the. administration of L-dopa which slows the progression of this disease in some humans. However, the neuropathological damage and the consequent behavioral deficits is not reversed by this treatment protocol.
Laboratory and clinical studies have shown that the transplantation of cells into the CNS is a potentially significant alternative therapeutic modality for neurodegenetative disorders such as Parkinson""s disease (Wictorin et al., 1990; Lindvall et al, 1990; Sanberg et al., 1994; Bjorlund and Stenevi, 1985; Freeman et al, 1994). In some cases, transplanted neural tissue can survive and form connections with the CNS of the recipient (i.e. the host). When successfully accepted by the host, the transplanted tissue (i.e. the graft) has been shown to ameliorate the behavioral deficits associated with the disorder (Wictorin et al, 1990). The obligatory step for the success of this kind of treatment is the prevention of graft rejection (i.e. graft acceptance).
Currently, fetal neural tissue is the primary graft source for neural transplantation (Lindvall et al., 1990; Bjorklund, 1992; Isacson et al., 1986; Sanberg et, al 1994). Other viabie graft sources inclcude adrenal chromaffin cells and various cell types that secrete nerve growth factors and trophic factors. The field of neural tissue transplantation as a productive treatment protocol for neurodegenerative disorders has received much attention resulting in its progression to clinical trials. Preliminary results and clinical observations are promising although the graft rejection phenomenon remains problematic.
Transplantation is also a valuable therapy for other diseases, such as insulin dependent diabetes mellitus. Insulin dependent diabetes mellitus is a major health problem. Current forms of therapy are not efficient and do not necessarily lead to a prevention of diabetic complications such as renal failure or blindness. A desirable treatment alternative is to provide the diabetic with an endogenous source of insulin, transplanting either the whole pancreas or the endocrine component of the pancreas (i.e. islets of Langerhans) into the diabetic recipient. Although, whole pancreas transplantation is successfully achieved with at least 60% of the grafts still functioning after transplantation for one year, a major weakness of this approach is the need for continuous immunosuppfessicn with powerful and toxic immunosuppressant drugs.
The transplantation of the isolated islets containing the insulin secreting xcex2-cells has received much attention in both animal models of diabetes (1-7) and in humans (8-16). However, islet transplantation to a variety of organ sites has met with little success as a viable treatment for diabetes. For example, islet transplantation of major histocompatibility complex (MHC) In the BB/W rat with spontaneous diabetes mellitus of autoimmune etiology results in destroyed islets within a few days by a recurrence of the autoimmune disease (17). Likewise, destruction of grafted cells in the diabeiic BB/W rat occurs in grafted islets of MHC-incompatibie donors (18, 19). In the course of finding a suitable organ or tissue site for islet transplantation, it was discovered that the relocated abdominal testis, in particular, provides an extraordinary safe environment for extended survival of islet grafts and some relief of the diabetic complications (20-22).
The testis has long been considered to be an immunologically privileged site (23-26) although the precise mechanism(s) by which it protects (suppresses) graft rejection has not been clearly defined. Isolated islets of MHC-compatible donors have been shown to survive for extended periods of time in the non-imminosuppressed BB/W rat if implanted in the rat""s testis which is then placed into the host""s abdominal cavity (20-22,27). Although the maintenance of functional islets allografts is significant, a more difficult task and far more potentially significant accomplishment, in terms of clinical applicability, is the induction of normoglycemia in diabetic animals by the implantation of cross-species islet xenografts.
Selawry and co-workers demonstrated the feasibility of such a procedure by successfully implanting incubated hamster islets into the BB/W rat abdominal testes (22,27,28). As a result of the abdominal testis/islet implant, the diabetic animals in these studies became normoglycemic. Long-term survival of the islet xenografts did not require prolonged immunosuppression to prevent rejection and to maintain normal sugar levels. In all cases implant viability required the protective milieu of the abdominal testis. It now appears that the donor origin of these isolated islets does not seem to influence their long-term survival. Islet cells grafted against major histocompatibility barriers (21), islet xenografts (27) and islets of MHC-compatible donors grafted into the testes of the diabetic BB/W rats functioned indefinitely in the recipient rendering the once diabetic animal normoglycemic.
The major weakness of this type of islet transplantation protocol is associated with the use of such an unconventional organ site. One major concern is the possibility of malignant transformation of germ cells at the higher core body temperature (29). More importantly, it would not be possible to use this transplantation protocol for the treatment of female diabetics.
Histological examination of grafted abdominal testes has shown that the islet implants are always found within the interstitial compartment of the gonad, which consists of the endocrine cells of Leydig, macrophages, blood vessels, testicular interstitial fluid and extracellular macromolecules (31). Any of the secretory products of these cells are potentially capable of inhibiting the immune response. For instance, Born and Wekerie (32, 33) showed that active suppression of immune responses occurred by Leydig cells in vitro. These investigators speculated that the Leydig cells might prevent lymphocyte proliferative responses by creating an xe2x80x9cimmunologically neutral zonexe2x80x9d around the seminiferous tubules and thus decreasing the danger of T-cell infiltration in to the intratubular spaces. It was shown by Williams (34) that leukemic cells accumulate in the interstitial compartment where they are apparently protected against destruction by the host""s immune defenses.
The xe2x80x9czone of protectionxe2x80x9d theory of Born and Wekerle (32) is attractive but it is not likely that this major component of the testicular interstitium, i.e. Leydig cells, is responsible for the synthesis of some protective (immunosuppressant) factor. Treatment of rats with ethane dimethanesulphonate (EDS), which selectively destroys the Leydig cell completely, including steroidogenesis and all other functions, had no adverse effects on the survival of intratesticular islet allografts (30). It is not probable that germ cells were involvedeither, since these cells are readily depleted in the abdominal testis. By eliminatin of these coils, Cameron and Sewiary concluded that the Sertoli cell was the most probable testicular cell type providing the testis with its unique immunologically privileged environment and that this cell was most likely responsible for the unexplained absence of islet rejection in abdominal testes (30). Based on these findings, Selawry and Cameron (35) attempted to create a similar immunologically privileged site outside of the testis utilizing Serioli cells as an immunosuppressant agent. To this end, isolated Sertoli cells were transplanted with isolated islets under the kidney capsule in female diabetic rats (see FIG. 1). Results from this study showed this novel transplantation protocol resulted in normoglycemia and that long-term islet allograft survival was achieved in a traditionally immunologically hostile site. We concluded that the Sertoli cell, independent of the testicular milieu, secreted an immunosuppressant factor(s) which was neither androgenic nor inhibitory to ovulation since 6 of the 7 mated recipients became pregnant, carried a pregnancy to term and nursed the pups successfully (35).
For the long-term treatment of diabetes, it is clear that the presence of viable Sertoli cells is a prerequisite for long-term islet graph success and maintenance of long-term beta cell function. We do not yet clearly understand, however, the mechanism(s) which yield this observation. The likely explanation is that the Sertoli cells secret an immunosuppressant factor(s) which cooperates with exogenous immunosuppressants such as cyclosporine A to prevent a complete immune response and subsequently tissue rejection (35). Sertoli cells are active secretory cell types synthesizing many proteins. some of which promote growth and others which have immunosuppressive capabilities (36, 55). Initial studies to verify such a factor have been positive to date. The effects of Sertoli cell conditioned media on Con A-stimulated spleen lymphocyte proliferation showed that products secreted by Sertoli cells inhibit lymphocyte proliferation in a dose-dependent manner. The synthesis was temperature dependent, occurrinry predominantly at 37xc2x0 C. and hormone dependent, requiring the presence of follicle stimulating hormone (FSH) in the Sertoli cell culture (see FIG. 2). We further examined the mechanism of inhibition of lymphocyte proliferation and showed that preconditioned Sertoli cell media inhibited the production of the lymphokine IL-2 in a dose-dependent manner (see FIG. 3A). Because the addition of exogenous IL-2 was not able to reverse this inhibition (see FIG. 3B), it appears likely that the preconditioned media inhibited both IL-2 production and T-lymphocyte responsiveness to IL-2 (38) in concurrence with similar finding by DeCesarts et al. (39) It is widely acknowledged that all proliferating T-cells express IL-2 receptors,. while resting cells do not, and that interaction of IL-2 with its receptor is an absolute requirement for the clonal expansion of activated T-cells (40). Because the prevention of IL-2 receptor interaction completely inhibits T-cell proliferation, we propose that both clonal expansion and viability of activated T-cells are suppressed by an immunosuppressive factor secreted by the Sertoli cells (35). In this fashion, the putative Sertoli cell derived immunosuppressant would appear to suppress the rejection by a mechanism similar to the action of cyclosporin A which also suppressed the production of IL-2 (41-44).
Although this hypothesis is appealing and with some research support of an indirect nature, it remains to be clearly unravelled. Recently, an additional and even more ppealing hypothesis has received consideration attention. Beligrau et al. (45*) in a letter to Nature showed that testis grafts that expressed Fas (CD95) ligand (FasL) survived indefinitely when transplanted under the kidney capsule, whereas testis grafts from gid mice (FasL deficient) were rejected when transplanted at the same site (45). A reverse transcriptase-polymerase chain reaction analysis demonstrated that Sertoli cells constitutively Pypress FasL mRNA. Additionally, they showed that isolated Sertoli cells derived from normal, but not the g/d mice survived indefinitely when transplanted under the kidney capsule. They concluded that the expression of functional FasL by Sertoli cells accounts for the immune-privilege nature of testis and suggested a mechanism by which Sertoli cells induce localized immune privilege to islets co-transplanted with Sertoli cells in an otherwise immune hostile site (i.e. subjacent the kidney capsule). They pointed out that FasL ligand-mediated immunosuppression would be expected to primarily target activated effector T cells rather than the activation steps that produce them, a mechanism by which Cyclosporin A produces immunosuppression. This would suggest that by targeting only activated T lymphocytes, grafted cell-associated FasL may provide a highly specific form of immunosuppression for ameliorating T-cell-dependent graft rejection. To this end, Lau et al, (46) transfected muscles cells with the FasL gene and co-transplanted them with islets beneath the kidney capsule and achieved local immunoprotection for the grafted islet, albeit for only 80 days. In a letter to Science, D Green declared this a stunning advance and declared that xe2x80x9cIt""s almost the Holy Grail of immunosuppression to restrict the suppression to the environment of the graftxe2x80x9d (47). Selawry and Cameron (35) achieved the same results with long-term imnmunoprotection of the grafted islets and long-term maintenance of normoglycemia in the diabetic rat by co-transplanting the islets with the natural producer of FasL, Sertoli cells. The salient features of terminally differentiated Sertoli cells that make them important and preferable as a transplantation facilitator are 1) they live for the life of the donor and may survive for the life of the recipient host (providing, thereby, long-term FAS-L induced local immunoprotection for the transplanted tissue or cells), 2) they do not divide and 3) they are easily isolated.
Since Sertoli cells secrete many growth enhancing factors including insulin-like growth factor I (55), the presence of Sertoli cells, in addition to their immunoprotective protective properties, may provide additional tropic and growth support to the transplant. Recently, Selawry et al, (48) showed that when cryopreserved pig Sertoli cells were thawed and immediately place in culture with Sertoli cells, there was a significant enhancement of post-thaw survival and insulin secretion when compared to thawed islets not co-cultured with Sertoli cells. They suggested that insulin-like growth factor I may have provided growth factor support to the cell membrane known to be damaged during freezing. Recently Sanberg et al (49-51) demonstrated that Sertoli cells can survive in the brain and, in fact, protect bovine adrenal chromaffin cell xenografts from rejection when co-transplanted into the striatum of the Parkinson""s disease rat model. Even more significant, Sertoli cells alone transplanted into the PD rat result in the amelioration of motion dysfunction to the same degree as do chromaffin cells indicating a type of successful growth factor therapy, as yet unknown, provided for by the transplanted Sertoli cells (52). Similar to islet cells, Carneron et al (53) have shown that the post-thaw viability of fetal brain cells is significantly enhanced if the neuron are co-cultured with Sertoli cells again indicating the generalized ability of Sertoli cell secretory products to support the viability of isolated cells. For both islets and neurons, the growth and viability enhancing characteristics of Sertoli cells were evident only when the Sertoli cells were present as opposed to only media soluble factors found in expended pre-conditioned Sertoli cell media.
The extra-testicular utilization of Sertoli cells in facilitated transplantation holds enormous potential based of the cell""s ability to provide for long-term localized immunosuppression and generalized growth enhancement of the transplanted cells and tissues. There is a distinct advantage to utilizing whole Sertoli cells rather than specific growth or immunosuppressant factors in that the Sertoli cell appears to continue expressing its desirable transplantation facilitation properties as long as the cell survives in the host, which may be for the life of the recipient. Because Sertoli cells cease mitotic activity following differentiation (54) and do not appear to re-acquire it following transplantation, it may be possible to transplant a stable population of Sertoli cells which remains stable for the life of the host. It is not an understatement to recognize that the utilization of extra-testicular Sertoli cells as transplantation facilitators opens the window to new and potentially significant protocols for transplantation success and represents the beginning xe2x80x9cof a new era in transplantationxe2x80x9d therapy (47).
In general, systematic. immunosuppression is necessary if successful transplantation is to be achieved in humans. Immunosuppression of the entire body (i e. systemic) can result, eventually, in graft acceptance. It is acquired, however, by placing the individual at medical risk making the immunosuppressant therapy itself more of a liability than a benefit in some cases. For a lack of a better immnosuppressant treatment, systemic immunosuppressants, with Cyclosporine-A (CsA) as the treatment choice, have been used as adjunctive therapy in neural transplantation protocols (Sanberg et al., 1994; Freeman et al., 1994; Borlongan et al., 1995). Arguably, systemic CsA treatment may be contraproductive to successful graft acceptance in the CNS because of its systemic effect and because CsA itself has been shown to cause detrimental side effects and may in fact, be cytotoxic to neural tissues (Berden et al., 1985; deGroen et al., 1984).
It would be useful to develop a mechanism that enhances the productive cell transplantation techniques already utilized for neurodegenerative disorders, such as Parkinson""s disease. This mechanism should improve these protocols in ways which would more effectively slow the neurodegenerative disease process, more actively promote the reestablishment of normal neural tissue physiology and better alleviate the functional disabilities associated with the neural tissue dysfunction. Likewise, it would be useful to provide trophic support for the transplanted cells. Further, it would useful if this support lead to the reduction or elimination of systemic immunosuppression while maintaining the ability to immunosuppress locally (i.e. at the graft site) by an immunosuppressant which is biologically tolerated by the host. Sertoli cells may provide this desired option since it is clear from the diabetic studies, as summarized above, that co-transplantation with Sertoli cells will deliver local immunosuppression and promote, therefore, efficient graft acceptance and functional restoration of the tissue-related dysfunction.
According to the present invention, there is provided a biological chamber including outer walls of Sertoli cells and an inner lumen. Also provided is a transplantation facilitator including a biochamber which is formed from an engineered Sertoli tissue construct. A method of making biochambers by co-culturing facilitator cells and therapeutic cells is also provided. Additionally, there is provided a method of transplanting cells by incorporating therapeutic cells into a biochamber and transplanting the biochamber containing the therapeutic cells. Further, a method of treatment using these engineered biochambers is also included.